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The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

Mitsudome Y, Takahama M, Hirose J, Yoshida N - AMB Express (2014)

Bottom Line: Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons.Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively.This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Applied Biosciences, University of Miyazaki, 1-1 Gakuen, Kibanadai-Nishi, Miyazaki 889-2192, Japan.

ABSTRACT
Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

No MeSH data available.


Related in: MedlinePlus

Gene silencing effects of Spo0A15 and 90 antisense oligonucleotides DNA on (A) spore formation and (B) dipicolinic acid accumulation inBacillus subtilisstr. 168. Values indicate the means ± standard deviation of three independent experiments.
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Figure 5: Gene silencing effects of Spo0A15 and 90 antisense oligonucleotides DNA on (A) spore formation and (B) dipicolinic acid accumulation inBacillus subtilisstr. 168. Values indicate the means ± standard deviation of three independent experiments.

Mentions: Spo0A is a signal protein involved in initiation of Bacillus sp. sporulation. Spo0A is activated by phosphorylation and serves as a translation regulation factor for one set of sporulation mRNAs (Errington, [1993]). Phosphorylation-mediated activation of spo0A is essential for switching of vegetative cells to spore cells, thus gene silencing of spo0A should cause repression of differentiation to spore cells. Spore formation rates of B. subtilis penetrons injected with antisense oligonucleotides (Spo0A15 or Spo0A90) was repressed to 24.4% and 35.8% of that of control B. subtilis penetrons respectively (Figure 5A). Alternatively, the amount of dipicolinic acid (Janssen et al. [1958]; Rotman and Fields [1968]) production of B. subtilis penetrons in which antisense oligonucleotide Spo0A15 and Spo0A90 was injected was repressed to 42.2 and 65.3% of control B. subtilis penetrons (Figure 5B). Presumably, these antisense DNAs bound to complementary sequences within the targeted spo0A mRNA and inhibited the translation of spo0A, thereby resulting in depression of spore formation in B. subtilis penetrons.


The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

Mitsudome Y, Takahama M, Hirose J, Yoshida N - AMB Express (2014)

Gene silencing effects of Spo0A15 and 90 antisense oligonucleotides DNA on (A) spore formation and (B) dipicolinic acid accumulation inBacillus subtilisstr. 168. Values indicate the means ± standard deviation of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230895&req=5

Figure 5: Gene silencing effects of Spo0A15 and 90 antisense oligonucleotides DNA on (A) spore formation and (B) dipicolinic acid accumulation inBacillus subtilisstr. 168. Values indicate the means ± standard deviation of three independent experiments.
Mentions: Spo0A is a signal protein involved in initiation of Bacillus sp. sporulation. Spo0A is activated by phosphorylation and serves as a translation regulation factor for one set of sporulation mRNAs (Errington, [1993]). Phosphorylation-mediated activation of spo0A is essential for switching of vegetative cells to spore cells, thus gene silencing of spo0A should cause repression of differentiation to spore cells. Spore formation rates of B. subtilis penetrons injected with antisense oligonucleotides (Spo0A15 or Spo0A90) was repressed to 24.4% and 35.8% of that of control B. subtilis penetrons respectively (Figure 5A). Alternatively, the amount of dipicolinic acid (Janssen et al. [1958]; Rotman and Fields [1968]) production of B. subtilis penetrons in which antisense oligonucleotide Spo0A15 and Spo0A90 was injected was repressed to 42.2 and 65.3% of control B. subtilis penetrons (Figure 5B). Presumably, these antisense DNAs bound to complementary sequences within the targeted spo0A mRNA and inhibited the translation of spo0A, thereby resulting in depression of spore formation in B. subtilis penetrons.

Bottom Line: Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons.Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively.This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Applied Biosciences, University of Miyazaki, 1-1 Gakuen, Kibanadai-Nishi, Miyazaki 889-2192, Japan.

ABSTRACT
Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

No MeSH data available.


Related in: MedlinePlus